**4. Enzymes**

All biological processes require presence of specific enzymes. Processes of reduction of protons as well as oxidation of hydrogen (see reaction below)require at least presence of three enzymes: iron hydrogenase, nickel-iron hydrogenase and nitrogenase.

$$\rm H\_2 \leftrightarrow 2H^\* + 2e^- \tag{1}$$

Hydrogenase exists in a number (~ 40) of prokariota, both aerobic and anaerobic, as well as in certain eukariota, e.g. in photosynthetic algae (Nickolet, 2000). Hydrogenases show different but significant sensitivity towards oxygen and light. Among more than 100 discovered hydrogenases essentially only those containing Fe and Ni atoms in active center are considered as the most attractive:


Active centers of both hydrogenases are composed from iron-sulfur clusters coordinated by carbonyl (CO) or cyanide (CN-) ligands.

**Iron hydrogenases** are the two directions enzymes because they catalyze both reduction of protons to the molecular hydrogen and the reverse reaction. There are three forms of these enzymes: monomeric – build only from the subunit controlling catalysis, dimeric, trimeric and tetrameric. Active centers located in these enzymes are not uniform either, however, all

*Rhodobacter spheroides* indicate strong chemotaxis with certain sugars, aminoacids and several organic acids (Packer, 2000). They are also capable to accept molecular nitrogen. Their metabolism is very elastic because they can germinate both in aerobic conditions (with

Under aerobic conditions this strain is used in purification of animal wastes (Huang, 2001) and biotransformation of toxins present in plant extracts (Yang, 2008). In the absence of oxygen *Rhodobacter spheroids* can be used in synthesis of carotenoides (Chen, 2006) and the

All biological processes require presence of specific enzymes. Processes of reduction of protons as well as oxidation of hydrogen (see reaction below)require at least presence of

H2 ↔ 2H+ + 2e-

Hydrogenase exists in a number (~ 40) of prokariota, both aerobic and anaerobic, as well as in certain eukariota, e.g. in photosynthetic algae (Nickolet, 2000). Hydrogenases show different but significant sensitivity towards oxygen and light. Among more than 100 discovered hydrogenases essentially only those containing Fe and Ni atoms in active center



Active centers of both hydrogenases are composed from iron-sulfur clusters coordinated by

**Iron hydrogenases** are the two directions enzymes because they catalyze both reduction of protons to the molecular hydrogen and the reverse reaction. There are three forms of these enzymes: monomeric – build only from the subunit controlling catalysis, dimeric, trimeric and tetrameric. Active centers located in these enzymes are not uniform either, however, all

inhibition but almost 100 times more active than [Ni-Fe] hydrogenases

towards hydrogen than [Fe] hydrogenase (Darensbourg , 2000).

(1)

Fig. 3. *Rhodobacter sphaeroides* ATCC 17039 (Garrity, 2005).

most of all in hydrogen generation (Kars, 2010).

are considered as the most attractive:

carbonyl (CO) or cyanide (CN-) ligands.

**4. Enzymes** 

or without light) as well as in anaerobic environment, in presence of light.

three enzymes: iron hydrogenase, nickel-iron hydrogenase and nitrogenase.

of them contain H-cluster (see Fig. 4) (Nicolet, 2000). Applying FTIR, EPR and XRD spectroscopy for analysis of monomeric hydrogenase, isolated from *Clostridium pasterianum* , it was found that H-cluster is composed from two basic units: [4Fe-4S] single group, responsible for electron transport, and the unique arrangement of [2Fe] capable to perform the reverse oxidation reaction of hydrogen. The regular cluster [4Fe-4S] is linked with four cysteine and sulfur atom of one of these forms the bridge bond between [4Fe-4S] and [2Fe]. In this dimeric system, the octahedral iron atoms are linked through two sulfur atoms (see Fig. 5) (Darensbourg, 2000). Moreover, it was found that these atoms are coordinated with five non-protein ligands (CO and CN-1) and water molecule. The bridge sulfur atoms forms additionally the 1,3- propanodithiol structure. The presence of covalent bond between sulfur atoms influence the charge of H-cluster and electric properties (Nicolet, 2000).

Fig. 4. Scheme of iron hydrogenase in *Desulfovibrio desulfuricans (Dd)* and *Clostridium pasterianum (Cp).* F – double cluster of [4Fe-4S], L-large subunit of H-cluster, S – small subunit of H cluster, Fd- [2Fe-2S] cluster related to ferredoxin. Pink color represents the unique structure of [4Fe-4S]. In *Dd* hydrogenase large and small subunits are connected via cysteine, whereas in Cp hydrogenase these units are linked with protein chain.

Fig. 5. Scheme of active center of iron hydrogenase (Darensbourg, 2000)

In active center of hydrogenase it is possible to identify such aminoacids as methionine and histidyne (Das, 2006). These two amino acids become attached to active center during formation of channels (for H2 and H+) connecting enzyme surface with reaction slit. The comparison of H-clusters in two strains of bacteria *Clostridium pasteurianum (Cp)* and *Desulfovibrio desulfuricans (Dd)* shows that in both cases the [2Fe] group is involved in hydrogen bond formation with lysine. However, when the second iron atom in *Cp* is

Microbiological Methods of Hydrogen Generation 231

because of the necessity of breaking the stable triple bond in nitrogen molecule and needs 16

Both components, nitrogenase and reductase are iron-sulphur proteins, in which iron is

*Reductase* (Figure 7) is a dimer with mass of 30 kDa composed of four iron atoms and four inorganic sulphides (4Fe-4S). The site for ATP/ADP bounding is located on the surface of this subunit. Reductase transfers electrons from the reduced ferredoxin towards dinitrogenase. This process occurs during hydrolysis of ATP with simultaneous dissociation

A. Red: ADP molecule obtained during ATP hydrolysis (location at the boundary of two dimers –

B. [4Fe-4S] cluster located on the boundary of dimers. Green – iron, orange - inorganic sulfur, black –

*Dinitrogenase* is a tetramer of the structure α2β2 and molecular weight of 240kDa (Figure 8). At the interface between the α and β subunits there is the P unit through which electrons are able to penetrate. Two cubo-octahedrons of 4Fe-4S are linked *via* sulphur atoms from cysteine residues. The flow electrons is realized from P unit to coenzyme Fe-Mo. This coenzyme is built of two units of M-3Fe-3S linked via sulphur atoms. In one unit M stands for Mo, while in the other one for Fe. Atmospheric nitrogen is transformed in the central part of coenzyme Fe-Mo. Multiple interactions of Fe-N type weaken the triple bond in molecular nitrogen which lowers the activation limit for nitrogen reduction (Berg, 2002). The synthesis of nitrogenase strongly depends on the light access to the medium and its intensity. Catalytic stability of nitrogenase is ensured by alternating light and dark 12-hour periods (day and night sequence) (Meyer, 1978). In the absence of molecular nitrogen and with large quantities of energy provided by ATP (Koku, 2002) nitrogenase catalyses hydrogen generation (see eq.3). Nitrogenase acts as a safety valve regulating cell reduction

carbon, yellow organic sulfur, blue – nitrogen, red – oxygen, Fig. 7. Reductase structure- nitrogenase component (Berg, 2002).

N2 + 8H+ + 8e + 16ATP → 2NH3 + H2 + 16ADP +16Pi (2)

ATP molecules per one molecule of nitrogen:

of the complex.

blue and yellow),

potential (Kars, 2010).

bonded with sulphur both in cysteine and the inorganic sulphide.

engaged with serine, in the case of *Dd,* alanine is involved instead. In the case of fermentative bacteria of the *Clostridium* family in the large unit of monomeric iron hydrogenase it was confirmed a presence of three excessive systems: the [2Fe-2S] structure, rarely existing [4Fe-4S] structure with slit and space constructed from two [4Fe-4S] systems (Vignais, 2006).

**Nickel–iron hydrogenase** isolated from *Desulfovibrio gigas* and *Desulfofibrio vulgaris* is composed from large subunit α (60 kDa) containing Ni-Fe active center and small subunits β (30 kDa) equipped with three iron-sulfur clusters. These clusters are involved in electron transfer between active centers, donors and acceptors. All these clusters are located in the strait lines in which [3Fe-4S] appears between two [4Fe-4S] structures (Vignais, 2006).

The active center f [Ni-Fe] hydrogenase exhibits the unique location of ligands (see Fig. 6)

Fig. 6. Scheme of active center of nickel-iron hydrogenase (Darensbourg, 2000).

Here, four molecules of cysteine coordinate one three valent nickel atom. Two of them coordinate simultaneously iron, also located in active center. This kind of arrangement induce formation of sulfur bridges between nickel and iron atoms. Moreover , non-protein ligands such as SO, CO, CN and CO, CN are located in active centers of *D. vulgaris* and *D. gigas*, respectively. Nickel and iron atoms are bonded with monoatomic sulfur (*D. vulgaris)* or oxygen (*D.gigas*) bridges. Generated space is an ideal place for hydrogen reduction with electrons transported by iron-sulfur clusters from the surface of enzyme. The change of nickel valance form III to 0 and the return to basic state together with reconstruction of sulfur (or oxygen) bridge is observed in this catalytic cycle.

**Nitrogenase** is considered as the essential part of nitrogen circulation system in the living world. Nitrogen present in the air, needs to be transformed into compounds acceptable by living organisms. The diazotrophic microorganisms, including the PNS bacteria, are able to transform atmospheric nitrogen into NH3. There three types of nitrogenases built of two separate protein units: dinitrogenase (either Mo-Fe protein, or V-Fe protein, or Fe-Fe protein) and reductase (Fe protein). The main task of reductase is the delivery of electrons of high reductive potential to nitrogenase which uses them to different reduce N2 to NH3. Six electrons are involved in this process to reduce the oxidation degree of nitrogen from 0 to 3. The enzyme also transfers two other extra electrons to protons with final formation of one molecule of H2. Reduction of nitrogen to ammonia is highly energy consuming process

engaged with serine, in the case of *Dd,* alanine is involved instead. In the case of fermentative bacteria of the *Clostridium* family in the large unit of monomeric iron hydrogenase it was confirmed a presence of three excessive systems: the [2Fe-2S] structure, rarely existing [4Fe-4S] structure with slit and space constructed from two [4Fe-4S] systems

**Nickel–iron hydrogenase** isolated from *Desulfovibrio gigas* and *Desulfofibrio vulgaris* is composed from large subunit α (60 kDa) containing Ni-Fe active center and small subunits β (30 kDa) equipped with three iron-sulfur clusters. These clusters are involved in electron transfer between active centers, donors and acceptors. All these clusters are located in the strait lines in which [3Fe-4S] appears between two [4Fe-4S] structures (Vignais, 2006).

The active center f [Ni-Fe] hydrogenase exhibits the unique location of ligands (see Fig. 6)

Fig. 6. Scheme of active center of nickel-iron hydrogenase (Darensbourg, 2000).

sulfur (or oxygen) bridge is observed in this catalytic cycle.

Here, four molecules of cysteine coordinate one three valent nickel atom. Two of them coordinate simultaneously iron, also located in active center. This kind of arrangement induce formation of sulfur bridges between nickel and iron atoms. Moreover , non-protein ligands such as SO, CO, CN and CO, CN are located in active centers of *D. vulgaris* and *D. gigas*, respectively. Nickel and iron atoms are bonded with monoatomic sulfur (*D. vulgaris)* or oxygen (*D.gigas*) bridges. Generated space is an ideal place for hydrogen reduction with electrons transported by iron-sulfur clusters from the surface of enzyme. The change of nickel valance form III to 0 and the return to basic state together with reconstruction of

**Nitrogenase** is considered as the essential part of nitrogen circulation system in the living world. Nitrogen present in the air, needs to be transformed into compounds acceptable by living organisms. The diazotrophic microorganisms, including the PNS bacteria, are able to transform atmospheric nitrogen into NH3. There three types of nitrogenases built of two separate protein units: dinitrogenase (either Mo-Fe protein, or V-Fe protein, or Fe-Fe protein) and reductase (Fe protein). The main task of reductase is the delivery of electrons of high reductive potential to nitrogenase which uses them to different reduce N2 to NH3. Six electrons are involved in this process to reduce the oxidation degree of nitrogen from 0 to 3. The enzyme also transfers two other extra electrons to protons with final formation of one molecule of H2. Reduction of nitrogen to ammonia is highly energy consuming process

(Vignais, 2006).

because of the necessity of breaking the stable triple bond in nitrogen molecule and needs 16 ATP molecules per one molecule of nitrogen:

$$\rm N\_2 + 8H^+ + 8e + 16ATP \to 2NH\_3 + H\_2 + 16ADP + 16P\_i \tag{2}$$

Both components, nitrogenase and reductase are iron-sulphur proteins, in which iron is bonded with sulphur both in cysteine and the inorganic sulphide.

*Reductase* (Figure 7) is a dimer with mass of 30 kDa composed of four iron atoms and four inorganic sulphides (4Fe-4S). The site for ATP/ADP bounding is located on the surface of this subunit. Reductase transfers electrons from the reduced ferredoxin towards dinitrogenase. This process occurs during hydrolysis of ATP with simultaneous dissociation of the complex.


Fig. 7. Reductase structure- nitrogenase component (Berg, 2002).

*Dinitrogenase* is a tetramer of the structure α2β2 and molecular weight of 240kDa (Figure 8). At the interface between the α and β subunits there is the P unit through which electrons are able to penetrate. Two cubo-octahedrons of 4Fe-4S are linked *via* sulphur atoms from cysteine residues. The flow electrons is realized from P unit to coenzyme Fe-Mo. This coenzyme is built of two units of M-3Fe-3S linked via sulphur atoms. In one unit M stands for Mo, while in the other one for Fe. Atmospheric nitrogen is transformed in the central part of coenzyme Fe-Mo. Multiple interactions of Fe-N type weaken the triple bond in molecular nitrogen which lowers the activation limit for nitrogen reduction (Berg, 2002). The synthesis of nitrogenase strongly depends on the light access to the medium and its intensity. Catalytic stability of nitrogenase is ensured by alternating light and dark 12-hour periods (day and night sequence) (Meyer, 1978). In the absence of molecular nitrogen and with large quantities of energy provided by ATP (Koku, 2002) nitrogenase catalyses hydrogen generation (see eq.3). Nitrogenase acts as a safety valve regulating cell reduction potential (Kars, 2010).

Microbiological Methods of Hydrogen Generation 233

initiated by one molecule of glucose, catalyzed by different enzymes and further transformed into 2 molecules of pyruvate. The energy liberated during oxidation of 3 phosphoglycerol aldehyde is sufficient for phosphorylation of generated acid towards 1,3 bisphosphoglycerol and reduction of NAD+ to NADH. This reaction is catalyzed by 3 phosphoglycerol dehydrogenase. Transformation of glucose to pyruvate is during glycolysis

Glucose is not the only substrate in glycolysis. Simple sugars such as fructose or galactose as well as complex sucrose – saccharose, lactose, maltose, cellobiose or cellulose can be used as the initial substrate for glycolysis. However, the incorporation of these complex sugars into

Glycerol can be considred as a good substarate for glycolysis. A part of glycerol is oxidized into dihydroxyacetone by glycerol dehydrogenase. Next, dihydroxyacetone is phosphorylated into phosphodihydroxyacetone in the presence of dihydroxyacetone kinase. Thanks to triozophosphate isomerase phosphodihydroxoacetone is transformed into 3-

There are known also other anaerobic pathways transforming glucose into pyruvate as e.g. Entner-Daudoroff or phosphate pentose pathway (Schlegel, 2003, Dabrock, 1992, Vardar-

**Entner-Doudoroff** pathway goes from glucose to pyruvate and is known also as 2-keto-3-detoxy-6-phosphogluconate. Here, glucose-6-phosphate is transformed with phosphogluconate dehydrogense into 6-phosphogluconate. In the next step, the removal of water from 6-phosphogluconate leads to formation of 2-keto-3-deoxy-6-phosphogluconate. This process is followed by formation of pyruvate and 3-phosphoglycol phosphate. These transformations are analogous to glycolitic pathway already described. One molecule of glucose is transformed into molecules of pyruvate with simultaneous formation of one NADPH (reduced dinucleotide nicotinoamine adenine phosphate and one molecule of ATP

**Pentophosphate pathway** is based on initial phosphorylation of glucose to glucose-6 phosphate with help of hexokinase. Further steps are more complicated. The glucose-6 phosphate dehydrogenaze transfer hydrogen to NAD simultaneously forming of gluconolactone. The phosphate gluconolactone dehydrogenase helps to generate 6 phosphogluconate acid. The last phase is based on decarboxylation of the acid into ribuloso-6 phosphate. The transfer of this compound into riboso-5-phosphate and xylulose-5-phosphate starts a non-oxidative phase. At this stage of reaction the reversible reaction between these compounds occurs with formation of sedoheptulose-7-phosphate and 3-phosphoglycerate aldehyde. Subsequent reactions can generate fructose-6-phosphate an erythrose-4-phosphate. In further reactions erythrose-4-phosphate is transformed into 3-phosphoglycerate aldehyde and fructose-6-phosphate. Thus, one cycle of pentophosphate pathway generates 2 molecules of fructose-6-phosphate, one molecule of 3-phosphateglycerol aldehyde, 3 molecules of CO2 and 6 molecules of NADPH. The pentophosphate pathway with glycolysis leads finally to the

In the next steps in anaerobic conditions, the oxidative decarboxylation of pyruvate occurs with acetylo-CoA and CO2 formation. This reaction is catalyzed by pyruvate oxyreductase

is accompanied by formation two molecules of ATP and two molecules of NADH.

glycolysis pathway require initial hydrolysis to the simple carbohydrates.

phosphoglycerol aldehyde and further participate in EMP pathway.

Schara, 2008, Chin, 2003).

(Schlegel, 2003).

pyruvate formation (Schlegel, 2003).

$$\rm 2H^{+} + 2e^{-} + 4ATP \rightarrow H\_{2} + 4ADP + 4P\_{i} \tag{3}$$

There are two main inhibitors of nitrogenase during hydrogen photobiogeneration: molecular oxygen and nitrogen. In the presence of molecular nitrogen occurs competitive nitrogen fixation reaction and this stops almost completely hydrogen evolution. Ammonium ions at concentrations higher than 20 μmol are successful but reversible inhibitors of hydrogen generation (Waligórska, 2009) as well. The nitrogen necessary for the cell functioning is usually provided by ethanolamine and glutamate.

A. Blue lines - two chains of α tetramer, yellow lines – β subunits, green – P group, red – Fe-Mo coenzyme.


Fig. 8. Dinitrogenase construction (Berg, 2002).

However, glutamate can be the source of nitrogen inhibiting hydrogen evolution similarly as non-ammonium compounds. It can when glutamate becomes the source of carbon after the other sources are exhausted (Koku, 2002). In order to avoid such situation a medium with a relatively high ratio of organic carbon to nitrogen should be applied (e.g. malate to glutamate =15/2 (Eroglu, 1999).
